title: Mechanisms of virus assembly probed by Raman spectroscopy: the icosahedral bacteriophage P22.
abstract: A microdialysis flow cell has been developed for time-resolved Raman spectroscopy of biological macromolecules and their assemblies. The flow cell permits collection of Raman spectra concurrent with the efflux of small solute molecules into a solution of macromolecules and facilitates real-time spectroscopic detection of structural transitions induced by the effluent. Additionally, the flow cell is well suited to the investigation of hydrogen-isotope exchange phenomena that can be exploited as dynamic probes of viral protein folding and solvent accessibility along the assembly pathway. Here, we describe the application of the Raman dynamic probe to the maturation of the icosahedral capsid of bacteriophage P22, a double-stranded DNA virus. The P22 virion is constructed from a capsid precursor (procapsid) consisting of 420 coat subunits (gp5) in an outer shell and a few hundred scaffolding subunits (gp8) within. Capsid maturation involves expulsion of scaffolding subunits coupled with shell expansion at the time of DNA packaging. Raman static and dynamic probes reveal that the scaffolding subunit is highly alpha-helical and highly thermolabile, and lacks a typical hydrophobic core. When bound within the procapsid, the alpha-helical fold of gp8 is thermostabilized; however, this stabilization confers no apparent protection against peptide NH-->ND exchange. A molten globule model is proposed for the native scaffolding subunit that functions in procapsid assembly. Accompanying capsid expansion, a small conformational change (alpha-helix-->beta-strand) is also observed in the coat subunit. Domain movement mediated by hinge bending is proposed as the mechanism of capsid expansion. On the basis of these results, a molecular model is proposed for assembly of the P22 procapsid.
authors.affiliation: Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri, Kansas City 64110, USA.
authors.forename: R
authors.initials: R
authors.lastname: Tuma
authors.forename: G J
authors.initials: GJ
authors.lastname: Thomas
mesh_terms: D017100:Bacteriophage P22 / Q000378:metabolism* / Q000502:physiology*; D002213:Capsid / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006361:Heating; D017551:Microdialysis; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012996:Solutions; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013816:Thermodynamics; D019065:Virus Assembly / Q000502:physiology*
chemical_list: D012996:Solutions